Recently, lots of active peptides have been discovered from hydrolysates of collagen and gelatin. A large number of reports on the use of the active peptide are available including use of Hyp-Gly for an inhibitor for arthritis or pressure ulcer (Patent Literature 1), use of Ala-Hyp, Leu-Hyp, and Ala-Hyp-Gly for a promoter for collagen synthesis (Patent Literature 2), use of peptides represented by Gly-X-Y-(Gly-Z-W) for an agent for inhibiting elevation glucose level in blood (Patent Literature 3), use of Gly-Pro-Ala-Gly for a DPP-4 inhibitor (Patent Literature 4), and application of Pro-Hyp's action of promoting fibroblast proliferation for cosmetics (Patent Literature 5). Further, with regard to digestion, absorption, and metabolism of collagen and gelatin when orally taken, it has been reported that dipeptides and tripeptides such as Pro-Hyp, Ala-Hyp, Leu-Hyp, Ala-Hyp-Gly, or Pro-Hyp-Gly are present in the blood (Non Patent Literature 1) and that Hyp-Gly is present in the blood (Non Patent Literature 2). Because these peptides are biologically active (Patent Literature 1, Patent Literature 2, and Patent Literature 5), it has been considered that the transfer of the dipeptide or tripeptide into the blood is one of the mechanisms for eliciting underlying its efficacy when collagen is orally taken. There are a large number of proteolytic enzymes that hydrolyze the collagens or gelatin; and studies have been conducted to efficiently generate biologically-active peptides by any of those enzymes or a combination thereof, and it has turned out that selection of the proteolytic enzyme is one of the critical factors.
Collagen has a unique amino acid sequence represented by -(Gly-amino acid X-amino acid Y)n- that glycine repeatedly appears every three residues and referred to as a so-called collagen-like sequence. To degrade the sequence to each unit, there are bacterial collagenases as proteolytic enzymes. Methods for degrading a collagen by using the bacterial collagenase are excellent to obtain Gly-X-Y type peptides. As reports on biological activities of the peptide generated by the bacterial collagenase, available are those on an agent for inhibiting elevation glucose level in blood using the above-mentioned peptide represented by Gly-X-Y-(Gly-Z-W) (Patent Literature 3), an agent for promoting collagen production using a tripeptide such as Gly-Ala-Arg (Patent Literature 6), and the like.
Meanwhile, there are methods of obtaining desired peptides by using plural proteolytic enzymes in combination, wherein the proteolytic enzymes are brought into reaction in plural steps. For instance, in order to produce a composition containing L-prolyl-L-hydroxyproline, there is a method which treats a collagen with a protease having the collagenase activity, thereafter uses an exopeptidase (Patent Literature 7). Further, available is a method for producing Glu-Hyp-Gly, Leu-Hyp-Gly, or Ser-Hyp-Gly which comprising a primary enzymatic treatment utilizing collagenase or protease derived from aspergillus flavus, and a subsequent secondary enzymatic treatment utilizing a peptidase that releases an amino acid from the N terminus of peptide having an amino acid other than proline or hydroxyproline at the second position from the N terminus to generate peptides that are not included in the products of the primary enzymatic treatment (Patent Literature 8).
Meanwhile, it has been known that zingibain is contained in ginger (Non Patent Literature 3), that collagen can be degraded by zingibain besides the bacterial collagenase and the like (Non Patent Literature 4), and that the protease activity of enzymes extracted from powder that has been obtained by grinding gingers rhizome in a polar solvent and drying the filtration residue is higher than the protease activity of enzymes extracted directly from the ginger rhizome (Non Patent Literature 5).
Further, it has been known that zingibain is a proline-specific cysteine protease and cleaves a peptide bond between the amino acid residue adjacent to Pro in the C-terminal side and the subsequent amino acid when the amino acid sequence is read from the N-terminal side (Patent Literature 9).